Adenylate cyclase toxin (ACT) is a fascinating bacterial virulence factor produced by Bordetella pertussis. It has unusual features, which make it an important prototype for study of the actions of several families of toxins. ACT is a 1706 amino acid protein that possesses an adenylate cyclase domain responsible for catalyzing the production of cAMP after it is delivered to the cytoplasm of the target cell. In addition, this molecule is a member of the RTX family of bacterial toxins and proteins, characterized by their calcium-binding repeats. Many of the family members are pore-forming hemolysins and cytotoxins and ACT is also hemolytic, apparently by formation of an oligomeric pore in the host cell membrane. In addition, this toxin elicits K+ efflux from host cells in a process that precedes and can be dissociated from oligomer formation. Work over the last several years has led to recognition that these activities of ACT are separate and distinct. In response, the objective of the next phase of this ongoing project is to understand the individual steps of toxin action and how they relate to one another. To accomplish this goal, the research will follow these Specific Aims: 1) physical characterization of ACT as it is prepared and evaluation of whether any or all of the activities are dependent on the formation of dimers by the toxin; 2) evaluation of the initial interaction of ACT with the target cell, using methods to define the early step before any membrane insertion is able to occur; 3) mapping to learn which portion of the toxin is inserted into the membrane, using electron paramagnetic resonance (EPR) spectroscopy, and determination of the relationship of that event to K+ efflux; 4) mapping of the portion of the catalytic domain which is delivered to the cytoplasm and investigation of whether intoxication and K+ efflux/hemolysis are mutually exclusive events for any single toxin molecule; and 5) characterization of the process of oligomerization and the domains of the toxin involved. This work is directed at understanding the mechanism of action of this toxin at a very basic level, with novel approaches and technologies. The results will be important for defining the role of this toxin in the pathogenesis of pertussis and for gaining better knowledge of its actions as a novel biomedical research probe and vehicle for delivery of foreign antigens to the host immune system. In addition, the information acquired from the proposed studies will contribute to the general field of toxin research, in which there are many examples of large extrinsic proteins gaining access to host cells by incompletely understood mechanisms.